Bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors for Abeta fibril formation

New bis-styrylpyridine and bis-styrylbenzene derivatives were designed and synthesized. These 34 compounds were evaluated by Abeta fibril formation inhibitory assay using thioflavin T as a dye (named ThT assay). Most of them showed excellent inhibitory activities for Abeta fibril formation at IC50 of 0.1-2.7 microM which is comparable to curcumin (IC50 of 0.8 microM). Among them, nine compounds were screened for their cytotoxicities on HT-22 cell by MTT assay at 1, 10, and 50 microM. In particular, I-7 and II-2 exhibited the best combination of inhibitory activity and compound cytotoxicity.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Thiazolidine derivatives as potent inhibitors specific for prolyl endopeptidase

A series of N-blocked L-proline-containing compounds and their derivatives were synthesized. Their inhibitory activities for prolyl endopeptidase from bovine brain were examined and compared with that of N-benzyloxycarbonyl-L-prolyl-L-prolinal, which is the most effective enzyme inhibitor hitherto reported. Introduction of a sulfur atom into pyrrolidine ring quite effectively increased the inhibitory activity: replacement of pyrrolidine with thiazolidine or thiazolidine aldehyde (thioprolinal) and conversion of L-proline to L-thioproline residue resulted in increase in the inhibitory activity. Thus, N-benzyloxycarbonyl-L-thioprolyl-thiazolidine (Z-Thiopro-thiazolidine) and Z-L-Thiopro-L-thioprolinal showed Ki values of 0.36 and 0.01 nM, respectively, for prolyl endopeptidase from bovine brain; both values were significantly lower than that of Z-Pro-prolinal (Ki, 3.7 nM).     

New betulinic acid derivatives as potent proteasome inhibitors

In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC(50) values of 1.42, 1.56, and 1.80 μM, respectively, which are three to fourfold more potent than the proteasome inhibition controls LLM-F and lactacystin.     

Novel dehydroabietylamine derivatives as potent inhibitors of acetylcholinesterase

Nowadays, the inhibition of acetylcholinesterase is one of the main pharmacological strategies for the treatment of Alzheimer’s disease. Therefore, a set of thirty-four derivatives of the diterpenoid dehydroabietylamine has been synthesized and screened in colorimetric Ellman’s assays to determine their ability to inhibit the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum). A systematic variation of the substitution of dehydroabietylamides enabled an approach to analogs showing a remarkable inhibition potency for AChE. Particularly N-benzoyldehydroabietylamines 11, 12 and 13 were excellent inhibitors for AChE, showing inhibition rates comparable to standard galantamine hydrobromide.     

New aryldithiolethione derivatives as potent histone deacetylase inhibitors

A series of dithiolethione derivatives was synthesized and the in vitro HDAC inhibitory activity was tested. The most active compounds, 1 and 2, exhibited an IC₅₀ in nM range with a strong hyperacetylation of histone H4 in A549 cells. The HDAC inhibitory activity comparable to that of SAHA and the inhibition of A549 cell proliferation suggest that these compounds are worthy of further studies as potential anticancer agents.     

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design,synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer’s disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.     

Search for dual function inhibitors for Alzheimer's disease: synthesis and biological activity of acetylcholinesterase inhibitors of pyridinium-type and their Abeta fibril formation inhibition capacity

Alzheimer's disease (AD) represents the most common neurodegenerative disorder, which is expressed through decline of mental function. Current treatment approaches include acetylcholinesterase inhibitors and NMDA-receptor partial antagonists. The most explored recent approaches that are closely related to the pathogenesis of this disease based on formally articulated amyloid hypothesis are: Abeta fibril formation inhibitors, amyloid precursor protein, and secretase inhibitors. [Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurol.2003, 2, 539] In view of the development of new AChE inhibitors as drugs capable of reducing the symptoms of AD, the capacity of newly synthesized AChE inhibitors of pyridinium-type to inhibit the AChE was examined and compared to those of other inhibitors of this type presented earlier. [Kapková, P.; Stiefl, N.; Sürig, U.; Engels, B.; Baumann, K.; Holzgrabe, U. Arch. Pharm. Pharm. Med. Chem.2003, 336, 523; Alptüzun, V.; Kapková, P.; Baumann, K.; Erciyas, E.; Holzgrabe, U. J. Pharm. Pharmacol.2003, 55, 1397] Furthermore, the anti-Abeta fibril formation property of AChE inhibitors of pyridinium- and bispyridinium-type was evaluated to expand their activity profile and to reveal potential additive pharmacological effects which may reinforce their therapeutic application besides their capacity of increasing acetylcholine levels. Abeta fibril formation studies were performed by means of thioflavin T fluorescence assay.     

Methanesulfonamido-cyclohexylamine derivatives of 2,4-diaminopyrimidine as potent ALK inhibitors

The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group into a methanesulfonamide. Tumor growth inhibition was observed when an orally bioavailable analog was evaluated in a Karpas-299 tumor xenograft mouse model.     

Discovery of quinolinone derivatives as potent FLT3 inhibitors

Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leukemia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC₅₀ of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC₅₀ greater than 10 μM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship.     

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.     

Dihydropyrimidine based hydrazine dihydrochloride derivatives as potent urease inhibitors

Four series of heterocyclic compounds 4-dihydropyrimidine-2-thiones 7–12 (series A), N,S-dimethyl-dihydropyrimidines 13–18 (series B), hydrazine derivatives of dihydropyrimidine 19–24 (series C), and tetrazolo dihydropyrimidine derivatives 25–30 (series D), were synthesized and evaluated for in vitro urease inhibitory activity. The series B–D were first time examined for urease inhibition. Series A and C were found to be significantly active with IC₅₀ values between 34.7–42.9 and 15.0–26.0 μM, respectively. The structure–activity relationship showed that the free S atom and hydrazine moiety are the key pharmacophores against urease enzyme. The kinetic studies of the active series A (7–12) and C (19–24) were carried out to determine their modes of inhibition and dissociation constants K_i. Compounds of series A (7–12) and series C (19–24) showed a mixed-type of inhibition with K_i values ranging between 15.76–25.66 and 14.63–29.42 μM, respectively. The molecular docking results showed that all the active compounds of both series have significant binding interactions with the active sites specially Ni-ion of the urease enzyme. Cytotoxicity of all series A–D was also evaluated against mammalian mouse fibroblast 3T3 cell lines, and no toxicity was observed in cellular model.     

Early synergy between Abeta42 and oxidatively damaged membranes in promoting amyloid fibril formation by Abeta40

Oxidative lipid membrane damage is known to promote the misfolding of Abeta42 into pathological beta structure. In fully developed senile plaques of Alzheimer's disease, however, it is the shorter and more soluble amyloid beta protein, Abeta40, that predominates. To investigate the role of oxidative membrane damage in the misfolding of Abeta40, we have examined its interaction with supported lipid monolayer membranes using internal reflection infrared spectroscopy. Oxidatively damaged lipids modestly increased Abeta40 accumulation, with adsorption kinetics and a conformation that are distinct from that of Abeta42. In stark contrast, pretreatment of oxidatively damaged monolayer membranes with Abeta42 vigorously promoted Abeta40 accumulation and misfolding. Pretreatment of saturated or undamaged membranes with Abeta42 had no such effect. Parallel studies of lipid bilayer vesicles using a dye binding assay to detect fibril formation and electron microscopy to examine morphology demonstrated that Abeta42 pretreatment of oxidatively damaged membranes promoted the formation of mature Abeta40 amyloid fibrils. We conclude that oxidative membrane damage and Abeta42 act synergistically at an early stage to promote fibril formation by Abeta40. This synergy could be detected within minutes using internal reflection spectroscopy, whereas a dye-binding assay required several days and much higher protein concentrations to demonstrate this synergy.     

Natural ortho-dihydroxyisoflavone derivatives from aged Korean fermented soybean paste as potent tyrosinase and melanin formation inhibitors

Natural o-dihydroxyisoflavone (ODI) derivatives with variable hydroxyl substituent at the aromatic ring of isoflavone and three known isoflavones were isolated from five-year-old Korean fermented soybean paste (Doenjang) and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells comparing with other known isoflavones, 7,8,4′-trihydroxyisoflavone (1) and 7,3′,4′-trihydroxyisoflavone (2) inhibited tyrosinase by 50% at a concentration of 11.21 ± 0.8 μM and 5.23 ± 0.6 μM (IC₅₀), respectively, whereas, 6,7,4′-trihydroxyisoflavone (3), daidzein (4), glycitein (5) and genistein (6) showed very low inhibition activity. Furthermore, those compounds significantly suppressed the cellular melanin formation by 50% at a concentration of 12.23 ± 0.7 μM (1), 7.83 ± 0.7 μM (2), and 57.83 ± 0.5(6) and show more activity than arbutin. But, compounds 3, 4, and 5 showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC₅₀ <40 nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains.     

Synthesis of novel indenoquinoxaline derivatives as potent α-glucosidase inhibitors

A series of new N-(11H-Indeno[1,2-b]quinoxalin-11-ylidene)benzohydrazide derivatives (3a–3p) were synthesized and evaluated for their α-glucosidase inhibitory activity. The synthesized compounds 3d, 3f, 3g, 3k, 3n, 3p and 4 showed significant α-glucosidase inhibitory activity as compared to acrabose, a standard drug used to treat type II diabetes. Structures of the synthesized compounds were determined by using FT-IR, ¹H NMR, ¹³C NMR, mass spectrometry and elemental analysis techniques.     

New racemosol derivatives as potent cyclooxygenase (COX) inhibitors

Racemosol (1) and 10-O-demethylracemosol (2), natural products from Bauhinia malabarica Roxb., exhibit potent in vitro anti-inflammatory activities against cyclooxygenase-1 and -2 (COX-1 and -2) enzymes. To investigate the structure-activity relationship (SAR) of these molecules, we prepared and fully characterized 17 derivatives by functionalizing one, two, or all three OH group(s) of 2 (Scheme). Both the size and polarity of the substituents as well as the substitution pattern in compounds 3a-q were found to be critical for anti-inflammatory activity. The orientation of the drugs and their mode of binding were studied by molecular docking based on the known 3D structure of the complex between COX-2 and the drug SC-558. Whereas the monoacetoxy derivative 3h exhibited an equally potent inhibitory activity towards both COX-1 and -2 (Table 1), its diacetoxy congener 3i was slightly more selective toward COX-2. In vivo anti-inflammatory tests showed that 3i and 2 are slightly more active than the reference compound phenylbutazone (Table 2).     

Novel pyrrolo-quinoline derivatives as potent inhibitors for PI3-kinase related kinases.

Several pyrrolo-quinoline gamma-lactones were found as novel inhibitors for two members of the PI3-kinase related kinase (PIKK) family, Ataxia-Telangiectasia-mutated (ATM) protein and the mammalian Target of Rapamycin (mTOR). Preliminary structure-activity relationship studies indicated that an electrophilic exocyclic double bond conjugated to the carbonyl group of the gamma-lactone ring was crucial for the PIKK inhibitory potency. One of the best ATM inhibitors in this series, DK8G557, showed IC(50) values of 0.6 and 7.0 microM for ATM and mTOR, respectively. This compound exhibited potent and selective growth inhibition activities in the NCI 60 human tumor cell line screen with a GI(50) MG-MID value of 2.69 microM. The best mTOR inhibitor in this series, HP9912, exhibited IC(50) values of 0.5 and 6.5 microM for mTOR and ATM, respectively. These compounds suggest novel leads for the discovery of potent small molecule inhibitors of PIKKs as potential anticancer drugs, with therapeutic activities as either single, or as sensitizing agents to conventional radio-, or chemo-therapeutic strategies.     

Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity

Beta-amyloid (Abeta) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Abeta responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Abeta aggregation still holds promise as a means to reduce Abeta neurotoxicity. In research presented here, we show that Hsp20, a novel alpha-crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Abeta amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Abeta of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Abeta fibril formation. Hsp20 was also able to significantly reduce Abeta toxicity to both SH-SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Abeta toxicity.     

Pyridazine and pyridazinone derivatives as potent and selective factor XIa inhibitors

Pyridazine and pyridazinone derivatives were designed and synthesized as coagulation factor XIa inhibitors. Potent and selective inhibitors with single digit nanomolar affinity for factor XIa were discovered. Selected inhibitors demonstrated moderate oral bioavailability.